PROJECT SUMMARY: To date, there are no drugs approved by the US Food and Drug Administration (FDA) for protection against cisplatin- or noise-induced hearing loss. We have developed an unbiased, phenotypic drug screen for protection against cisplatin ototoxicity, using a cell line derived from neonatal mouse cochleae. From a library of 4,359 unique compounds, including 844 FDA-approved drugs, we have identified a top hit that exerts strong protection against cisplatin ototoxicity in the cochlear cell line and cochlear explant culture, with an excellent therapeutic index (>200) and an IC50 of ~150 nM. We have confirmed that when locally delivered to adult mice via injection through the eardrum, this top hit protects against cisplatin and noise injury. Therefore, we have a validated top hit that protects against cisplatin- and noise-induced hearing loss. Here we propose to optimize our top hit to generate a lead compound by improving local delivery methods and by performing sequential analyses, including medicinal chemistry, structure-activity relationship (SAR), absorption, distribution, metabolism, excretion, safety/toxicology (ADMET), and in vivo pharmacokinetics (PK) and pharmacodynamics (PD) studies. Our top hit and its analogs, after testing in our streamlined assays, will serve as candidate compounds for preclinical and clinical studies of their safety and efficacy when locally delivered for treatment of hearing loss.